effect of three days storage of coated spermatozoa at cooling and adding seminal plasma on ram fertility

introduction artificial insemination (ai) has only been used as a supplement to natural mating. ai, when used in conjunction with accurate progeny testing schemes, can substantially increase the rate of genetic progress compared with that of natural service. moreover, the use of ai causes the limitation of the transmitted diseases. cervical insemination with frozen-thawed ram semen has not been widely adopted, probably because of the relative poor fertility obtained. thus using fresh and diluted semen is only approach for performing ai. ai is currently limited by the poor fertility achieved after cervical insemination with the storage of liquid semen at sub-ambient temperature. the success of this procedure in sheep is restricted by the short length of time that ram sperm can be stored in a liquid state. moreover, the effect of cooling on sperm differs depending on species. it is also well known that ram spermatozoa are more sensitive to cold-shock stress than those of other species. seminal plasma, as physiological secretion, is a complex mixture of secretions originating from testis, epididymis and accessory sex glands which is mixed with epididymal sperm at ejaculation; it serves as the carrier of sperm to the female genital tract. this mixture contains numerous factors such as organic and nonorganic material which play an important role in the final maturation of the spermatozoa through hormonal, enzymatic and surface-modifying events. during natural mating, a mechanism may be activated to separate spermatozoa from seminal plasma. after being ejaculated into the vagina, sperm swim through cervical mucus and enter the uterus within minutes (>30 min); cervical mucus acts as a barrier for seminal plasma. in the artificial insemination industry, seminal plasma with all the useful and harmful components is not removed from semen and is in contact with sperm throughout cooling, freezing and storage. on the other hand, it was demonstrated that the auto-destructive activity of seminal plasma was decreased which may be reduced by coating spermatozoa for less than 5 min during collection with the commercial diluent supplemented with egg yolk. the detrimental effect of lipid efflux induced by seminal plasma may be abolished by decreasing the time of the contact between seminal plasma and sperm. the objective of this study was to determine whether coating method, as a collection method, can improve fertility of ram spermatozoa after 72 h storage. materials and methods experiment was conducted to evaluate the effect of seminal plasma on coated spermatozoa fertility by using 111 ewes, aged between 1 and 3 years. semen from four mature, healthy and fertile thaleshi rams, aged between 2 and 5 years, were used for ai. the animals were housed at the faculty of agricultural sciences, education research and practice farm, university of guilan, south of rasht (it is located at 37° 12´ north latitude and 49° 39´ east longitude) and fed daily with alfalfa hay and 0.5 kg of concentrate, and provided salt lick and water ad libitum. semen was collected throughout the breeding season (august, 2011) by using an artificial vagina. ejaculates from each ram were collected in a tube containing 5 ml of coating medium (269 mm tris (hydroxymethy1) aminomethane, 52 mm d-fructose, 89 mm citric acid, 2000 iu/ml penicillin g and 0.4 mg/ml streptomycin ph=7.0) at72 h before insemination. two or three consecutive ejaculates fromeach ram were collected. the ejaculates were placed in a water bath (35○c) immediately after collection. semen quality was assessed, and to be accepted as a donor, and the ejaculation of each ram ejaculation had to fulfill the following demands concerning semen quality: volume ≥ 0.5 ml, macroscopic good visual mass activity (sperm motility ≥ 75%), sperm concentration ≥ 3 × 109⁄ml and normal sperm morphology ≥ 90%. coated ejaculates were centrifuged for 10 min at 700 × g at room temperature and the supernatant was removed. the pellets were diluted by tris-glucose up to 800 × 106 sperm/ml then they were split into three parts (e-s+, e-s-and e+s-) and incubated at 5 c. after 68 h‚ samples were centrifuged by 700 × g 10 min at 5 °c. in e-s+, supernatant was removed and added 10% crude seminal plasma. in e-s-‚ supernatant was removed and added tris-glucose. in e+s-‚ pellet was mixed with supernatant. samples were packaged into straws‚ incubated at 5 °c for 4 h and inseminated 72 h after collection. ewes were allocated to three groups and inseminated after synchronizing estrus by using cider (14 d) and injection hcg (400 iu). results and discussion the results showed that the lambing rate was higher in ewes of second parity (18.91%) than ewes of first parity (5.12%). there was no significant difference between e-s- (24.32%) and e+s- (10.81%) although the percentage of lambing rate was higher about 10 % in e-s- than e+s-. there was no significant difference between e-s+(5.12%) and e+s- on lambing rate. the pair-wise comparison of the lambing rates between the three groups showed significant higher results for e-s- compared with e-s+. therefore, fertility of coated spermatozoa was not improved by adding 10% crude seminal plasma after three days storage at 5 c.